Heterocycles 27. Microwave assisted synthesis and antitumour activity of novel phenothiazinyl-thiazolyl-hydrazine derivatives

Article abstract of DOI:10.1002/ardp.201100355

A series of new phenothiazinyl-thiazolyl-hydrazine derivatives were synthesized by Hantzsch cyclization of 1-(10-ethyl-10H-phenothiazin-3-yl)- methylidene-thiosemicarbazide with α-halocarbonyl derivatives. Comparison between classical and microwave assisted synthesis emphasizes the great advantages induced by microwaves irradiation which afforded high reaction yields in much shorter reaction time. Structural assignments were based on spectroscopic methods (high resolution NMR, FTIR, MS). The new compounds were tested in vitro for their antiproliferative activity against tumor cell lines using spectrometric methods. Most of the compounds exhibit cytotoxicity against hepatic and colon tumor cells in a dose-dependent mode and a relationship between the structure and their biological activity was observed. Microwave assisted synthesis afforded excellent yields of new phenothiazinyl-thiazolyl- hydrazine derivatives. Their cytotoxicity was tested in vitro based on spectrometric methods. Copyright

Full text of DOI:10.1002/ardp.201100355

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
1
Full Paper
Heterocycles 27. Microwave Assisted Synthesis and
Antitumour Activity of Novel Phenothiazinyl-Thiazolyl-
Hydrazine Derivatives
Adriana Ignat1,2, Tamas Lovasz2, Mihai Vasilescu3, Eva Fischer-Fodor4, Corina Bianca Tatomir4,
Castelia Cristea2, Lumini¸ta Silaghi-Dumitrescu2, and Valentin Zaharia1
1 Faculty of Pharmacy, ‘‘Iuliu Ha¸tieganu’’ University of Medicine and Pharmacy, Cluj-Napoca, Romania
2 Faculty of Chemistry and Chemical Engineering, ‘‘Babes Bolyai’’ University RO-400028, Cluj-Napoca,
Romania
3 Faculty of Physics, ‘‘Babes¸ Bolyai’’ University, Cluj-Napoca, Romania
4 Radiobiology and TumorBiology Laboratory, I. Chiricuta Cancer Institute, Comprehensive Cancer Center,
Cluj-Napoca, Romania
A series of new phenothiazinyl-thiazolyl-hydrazine derivatives were synthesized by Hantzsch
cyclization
of
1-(10-ethyl-10H-phenothiazin-3-yl)-methylidene-thiosemicarbazide
with
a-
halocarbonyl derivatives. Comparison between classical and microwave assisted synthesis
emphasizes the great advantages induced by microwaves irradiation which afforded high reaction
yields in much shorter reaction time. Structural assignments were based on spectroscopic methods
(high resolution NMR, FTIR, MS). The new compounds were tested in vitro for their antiproliferative
activity against tumor cell lines using spectrometric methods. Most of the compounds exhibit
cytotoxicity against hepatic and colon tumor cells in a dose-dependent mode and a relationship
between the structure and their biological activity was observed.
Keywords: Cytotoxicity / Hepatocarcinoma / Hydrazine / Microwave assisted synthesis / Phenothiazine / Thiazole
Received: October 6, 2011; Revised: January 17, 2012; Accepted: February 16, 2012
DOI 10.1002/ardp.201100355
Introduction
zine derivatives induced antiproliferative effects on multi-
drug-resistant cancer cells [16, 17] and exhibited a synergic
effect in combination with some antineoplastic drugs.
Our target was to obtain new compounds with antiproli-
ferative activity against tumor cell lines, by assembling the
hydrazino-thiazolyl group and the phenothiazine nucleus in
the same molecular structure, encouraged by our previous
results in the synthesis and evaluation of biological activity of
some hydrazinothiazoles [18], sulphonyl-hydrazinothiazoles
[19], aroyl-hydrazinothiazoles used as intermediates in the
synthesis of thiazolo[2,3-c][1,2,4]triazoles [20, 21] and p-tolu-
enesulphonyl-hydrazinothiazoles [22]. For the optimization
of the reaction conditions and an ecofriendly synthetic
approached in the synthesis of the new phenothiazinyl-
thiazolyl-hydrazine derivatives, our goal was to test the
Compounds containing hydrazino-thiazole pharmacophore
group may develop antimicrobial [1, 2], antifungal [2], anti-
inflammatory [3] or antioxidative [4] activity. Some synthetic
hydrazino-thiazole derivatives also exhibited antitumour
activity [5–8].
Phenothiazine derivatives are well known due to their
widespread therapeutic applications [9, 10], but more
recently, their cytotoxic potential was also investigated
and certain phenothiazine derivatives were found to be effec-
tive against ovarian cancer cells [11], human cervical cancer
cells [12], lymphomas and COLO 320 cells [13] and were
proved to induce apoptosis in neuroblastoma, glioma [14]
and lung fibroblast [15] cell lines. Moreover, some phenothia-
Additional correspondence: Valentin Zaharia, Faculty of Pharmacy,
‘‘Iuliu Ha¸tieganu’’ University of Medicine and Pharmacy, RO-400012, Victor
Babes 41, Cluj-Napoca, Romania.
E-mail: vzaharia@umfcluj.ro
Fax: 0040 264 597257
Correspondence: Castelia Cristea, Faculty of Chemistry and Chemical
Engineering, RO-400028, 11 Arany Janos street, Cluj-Napoca, Romania.
E-mail: castelia@chem.ubbcluj.ro
Fax: 0040 264 590818
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Table 1. Optimization of the microwavea) assisted synthesis of
thiosemicarbazone 1
possibility of using the microwave assisted synthesis, a tech-
nique which, applied in the preparation of various hetero-
cycles including phenothiazine [23–25] and thiazole [26–28],
showed major advantages such as enhanced reaction rates,
easy workup of the reaction mixture and ensured high yields
of products. The new phenothiazinyl-thiazolyl-hydrazine
derivatives thus obtained gave promising results by in vitro
test of antiproliferative activity against hepatic and colon
tumor cell lines, as presented below.
Entry
Solvent
Reaction
time (min.)
Temperature
(8C)
Yield
(%)
1
2
3
4
5
6
Ethanol
Ethanol
Ethanol
30
20
10
10
20
20
100
100
100
80
100
100
95
65
48
61
51
5
Ethanol
Ethanol/Water
Water
Results and discussion
a)
Power P ¼ 200 W.
Chemical synthesis
The strategy applied for the chemical synthesis of the target
heterocyclic hydrazine derivatives 2a–g containing thiazole
and phenothiazine units in the same molecular structure, is
based on two reaction steps: (i) the condensation of 10-alkyl-
10H-phenothiazine-3-carbaldehyde with thiosemicarbazide,
followed by (ii) the Hantzsch thiazole synthesis using several
a-haloketones. In order to find the most suitable reaction
conditions which generate 1-((10-alkyl-10H-phenothiazin-3-
yl)methylidene)thiosemicarbazide 1 [29–31] and also for a
more ecofriendly synthetic approach, the syntheses were
performed both under classical convective and microwave
assisted heating techniques (Scheme 1).
a-halocarbonyl derivatives (such as chloroacetone, 1,3-
dichloroacetone, a-bromoacetophenone, 3-chloroacetylace-
tone, ethyl-a-bromoacetylacetate, ethyl-g-bromoacetylace-
tate, ethyl-bromopyruvate), as shown in Scheme 2. Acetyl-
hydrazides 3a–h were obtained in high yields by stirring 2a–g
with acetic anhydride for 15 min at reflux, in the presence of
catalytic amounts of pyridine (Scheme 2).
The target phenothiazyl-thiazolyl-hydrazine derivatives
2a–g were obtained in moderate yields by the classical
Hantzsch cyclization at room temperature, but excellent reac-
tion yields were observed when microwave irradiation was
applied (Table 2). Optimal reaction conditions for the micro-
wave assisted Hantzsch cyclization shown in Table 2 method B,
were established after several experiments of irradiation at
different reaction temperatures (40 or 608C) and times (30, 60
or 90 min) in the presence of dimethylformamide solvent.
Very high yields of each compound 2a–g were obtained by
microwave assisted synthesis after 90 min at 608C.
The comparison between classical and microwave assisted
reaction conditions presented in Table 2 shows that Hantzsch
cyclization afforded only 43–68% yields of target compounds
2a–b after a very long reaction time (24 h) at room tempera-
ture, while important improvements of the reactions yields
(89–99%) were observed when microwave irradiation was
applied for much shorter reaction times (1.5 h), thus recom-
mending the microwave assisted synthesis as the most suit-
able technique for obtaining the phenothiazinyl-thiazolyl-
hydrazine derivatives 2a–g.
Under classical convective heating to reflux in ethanol, the
condensation of 10-ethyl-10H-phenothiazine-3-carbaldehyde
with thiosemicarbazide required 4 h reaction time in order
to obtain good yields (87%) of thiosemicarbazone 1.
Alternative conditions were applied during the optimiz-
ation of the microwave assisted synthesis of 1, as presented in
Table 1. Good to excellent yields were obtained by dielectric
heating in the presence of ethanol solvent (Table 1, entries
1–3) in significantly shorter reaction time, as compared to
the convective heating technique. For a greener synthesis, we
attempted to replace the organic solvent with water, but
unfortunately in such conditions 1 was recovered only in
traces (Table 1, entry 6). Moderate yields of 1 can be obtained
in ethanol/water mixture (Table 1, entry 5), thus indicating
the possibility of using water at most as a co-solvent.
The phenothiazinyl-thiazolyl-hydrazine derivatives 2a–g
were obtained by both classical and microwave assisted
Hantzsch cyclization of thiosemicarbazone 1 with several
The structures of the obtained compounds 1, 2a–g, 3a–g
were assigned based on their recorded high resolution
CH2 CH3
N
CH2 CH3
N
A or B
+
NH2 NH
C
S
NH2
CH
N
NH
C
S
NH2
CHO
S
1
S
Scheme 1. Condensation of 10-ethyl-10H-phenothiazine-3-carbaldehyde with thiosemicarbazide. Reaction conditions: A) abs. ethanol,
788C, convective heating; B) abs. ethanol or ethanol/water mixture, 1008C, MW irradiation.
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Novel Phenothiazinyl-Thiazolyl-Hydrazine Derivatives
3
CH2 CH3
N
CH
N
NH
R1
C
S
NH2
S
1
O
+
X
C
a
R1
R2
-H
-H
-H
-CO-CH3
-COO-C2H5
CH
Compound
2a,3a
R2
-CH3
-CH2Cl
-C6H5
-CH3
-CH3
CH2 CH3
N
2b,3b
2c,3c
2d,3d
2e,3e
R1
N
CH
N NH
R2
S
S
2f,3f
2g,3g
-CH2-COO-C2H5 -H
-H -H
_
2a g
+ (CH CO)2O
b
3
CH2 CH3
N
Scheme 2. Synthesis of phenothiazyl-thiazol-
yl-hydrazine derivatives 2a–g and acetyl deri-
vatives 3a–g. (a) Hantzsch cyclization, DMF,
ethanol, reaction conditions as given in
Table 2. (b) Acetic anhydride, pyridine (catalytic
amount), reflux 15 min.
R1
N
CH
N
N
R2
S
S
CO
CH3
_
3a g
Table 2. Experimental conditions for Hantzsch cyclization applied in the synthesis of phenothiazinyl-thiazolyl-hydrazine derivatives 2a–g
Compound Methoda)
Temperature
(8C)
Time
(min)
Pressure
(bar)
Power
(W)
Yield
(%)
2a
2b
2c
2d
2e
2f
A
B
A
B
A
B
A
B
A
B
A
B
A
B
25
60
25
60
25
60
25
60
25
60
25
60
25
60
1440
90
1440
90
1440
90
1440
90
1440
90
1440
90
1440
90
1
1.7
1
1.7
1
1.7
1
1.7
1
1.7
1
1.7
1
1.7
–
100
–
100
–
100
–
100
–
100
–
100
–
57
98
43
92
62
99
63
89
60
92
68
94
59
91
2g
100
a)
A, classical conditions; B, microwave irradiation.
1H NMR, 13C NMR, MS and FT-IR spectra. In the 1H NMR
spectra, the heteroaromatic rings and the carbonyl substitu-
ents determine characteristic deshielding effects which can
be observed on key signals such as for example the signal of
Biological activity
The mathematic parameter used to quantify the compounds
antiproliferative effect against the tumor cells was the half
maximal inhibitory concentration (IC50). These data were
obtained by generating dose–response curves for every com-
pound using the biostatistics software. The values IC50
obtained for each compound tested for both HepG2 and
CC531S cell lines are shown in Table 3.
–
the proton in the azomethine group (–CH N–) which appears
–
as a singlet situated at 7.68 ppm in the thiosemicarbazone 1,
7.76 ppm in the phenothiazinyl-thiazolyl-hydrazine 2a and,
respectively 8.21 ppm in the corresponding acetyl-derivative
3a. Complete assignment of the structures was based on 2D
homo- and heteronuclear correlation NMR experiments
COSY, HMQC and HMBC.
The hepatic HepG2 tumor cell line growth was inhibited by
all compounds 1, 2a–f and 3b–g (Fig. 1b). We compared the
treated cells proliferation with the untreated, control tumor
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Table 3. In vitro antiproliferative activity of phenothiazinyl-thiazolyl-hydrazine derivatives against hepatic HepG2 tumor cells and colon
carcinoma CC531S cells (determined by colorimetric MTT method).
Cpd
HepG2 cells IC50 mg/mL
CC531S cells IC50 mg/mL
1
2a
2b
2c
2d
2e
2f
3b
3c
3d
3e
3g
5.64
2.40
12.72
29.87
24.95
4.25
2.78
1.83
3.84
7.92
3.42
4.22
8.64
37.39
19.58
4.74
2.21
2.60
2.98
3.67
8.04
31.75
14.72
6.77
3.26
2.57
4.21
4.36
7.03
3.61
9.88
10.94
8.18
16.84
6.01
9.00
13.28
9.86
2.60
6.10
7.01
9.15
4.12
7.18
16.61
9.42
20.12
13.49
9.35
8.45
5.00
10.80
6.77
14.75
12.44
10.27
13.34
8.18
11.62
21.15
5.50
4.93
4.77
14.14
11.65
13.34
2.12
13.49
10.28
13.19
2.65
14.7
16.35
2.39
9.82
9.27
1.638
Cisplatin
1.87
Cl of discrepancy, two-tailed p value <0.0395 for each com-
pound). ANOVA one-way analysis of variance and Dunnett’s
multiple comparison test indicate that the activity of com-
pounds 1, 2a, 2e, 2f, 3b and 3c is comparable to cisplatin
cytotoxicity, while the activity of 2b, 2c, 2d, 3d, 3e and 3g is
significantly lower (p <0.05, p value extremely significant).
The colon carcinoma CC531S cells proliferation was better
inhibited by compounds 1, 2a and 3c (Fig. 1a). According to
the two-tailed t-test and the ANOVA analysis the cytotoxicity
of 1, 2a–f and 3b–g is in each case significant relatively to the
untreated cells (95% confidence interval, two-tailed p value
not higher than 0.043). The biological effect of 1, 2a, 3c, and
3e is similar to cisplatin, while for the other compounds the
activity shows significant differences (p <0.05). Within this
group, the compounds fall in two categories: 2c, 3d, 2f and 2d
with cytotoxicities which differ slightly from the chemother-
apy drug (Dunnett’s multiple comparison test, p value sum-
mary significant), and 2b, 3b, 2e and 3g with significantly
lower activity (p value summary extremely significant).
The comparison of the cytotoxicity of compounds 2b–e
with their acetylated analogues 3b–e upon HepG2 cell line
show that the presence of the acetyl group causes a decrease
of IC50 value as compared to unacetylated parent compound,
except for 3e, which exhibits a larger IC50 value. The cytotox-
icities of compounds 2b and 3b differ significantly (Wilcoxon
matched pair test, two-tailed p value 0.0074, very significant),
the difference between the activities of 2c and 3c is extremely
significant (p ¼ 0.0002), in the pair 2e–3e there is a signifi-
cant difference between IC50 values (p ¼ 0.0105), while the
activities of 2d and 3d do not diverge significantly.
Figure 1. Cytotoxic activity of phenothiazinyl-thiazolyl-hydrazine
derivatives quantified by IC50 values upon (a) CC531S cell lines
(b) HepG2 cell lines.
cells. As positive reference values we determined the IC50
values of cisplatin, a commonly used chemotherapy drug,
which showed effectiveness in tumor cell inhibition, and was
proven to be active against HepG2 [32, 33] and CC531S [34]
cells. Cytotoxicity upon cells treated with compounds 1, 2g
and 3g was significant in each case; Graph Pad Prism column
statistics two-tailed t-test indicates significant inhibition (95%
The comparison of the cytotoxic activity of compounds
2b–e with their acetylated analogues 3b–e and the statistic
analysis showed that against the CC531S cell line there are no
notable differences between 2b and 3b, 2d and 3d (Wilcoxon
matched pair test, Spearman two-tailed p value summary not
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Novel Phenothiazinyl-Thiazolyl-Hydrazine Derivatives
5
significant), while for the pairs 2c–3c and 2e–3e the IC50
values differ significantly (p <0.05). The presence of the ace-
tyl group in the molecule improved significantly the anti-
proliferative potential in compounds 3c and 3e.
The presence of acetyl group in 3e gives a divergent out-
come; the compound is more cytotoxic against colorectal
tumor cells, but less toxic against hepatic cells as compared
to its non-acetylated parent 2e.
hepatic and colon carcinoma cells. A structure–activity
relationship survey of phenothiazinyl-thiazolyl-hydrazine
derivatives shows that the presence of substituents such as
methyl or chloromethyl in position 4 of the thiazole ring
enhanced the cytotoxic potential of the compounds, while
the steric hindrance induced by the presence of bulk sub-
stituents in position 4 and 5 of the thiazole moiety weakened
the antiproliferative activity. The acetylation of hydrazone
unit does not affect significantly the antiproliferative effect,
but slightly enhances the cytotoxicity against HepG2 and
CC531S cell lines. Among the studied compounds 2a, 2b,
2f, 3b, 3c and 3e exhibit a distinct therapeutical potential.
The cytotoxicity of the new compounds against the color-
ectal cells; is higher as compared to the cytotoxicity against
hepatic cells (two-way ANOVA test, Bonferroni post-test,
p <0.0001).
The two groups of acetylated (3b, 3c, 3d, 3e, 3g) and non-
acetylated (2a, 2b, 2c, 2d, 2e) phenothiazinyl-thiazolyl-hydra- Experimental
zine analogues were analyzed using grouped statistics; the
two-way ANOVA test indicates that IC50 values for non-acetyl-
ated compounds are significantly higher for the HepG2 liver
cells (Bonferroni post test, p <0.01), while in the case of
CC531S cell line the differences are irrelevant between the
two groups (p >0.05).
Chemistry
Melting points were determined with an Electrothermal IA 9200
digital melting point apparatus and are uncorrected. Elemental
analyses were performed by a Vario EL III instrument. The mass
spectra were recorded with a Shimadzu QP 2010 EI mass spec-
trometer. IR spectra were recorded on a Bruker Vector 22 FT-IR
spectrometer fitted with a Golden Gate ATR accessory. 1H and
13C NMR spectra were recorded in CHCl3-d1 or DMSO-d6, in 5 mm
tubes at RT, on a WM-400 MHz Bruker NMR spectrometer.
Assignments were made based on standard 2D HSQC and
HMBC techniques. The spectral data are listed in the experimen-
tal part. Microwave assisted syntheses were performed in sealed
vessels using a CEM Discover LabMate instrument, insured with
on-line inside temperature and pressure control.
The biological activity of phenothiazinyl-thiazolyl-hydra-
zine derivatives slightly varies according to the substituent
attached in positions 4, 5 of the thiazole ring and acetylation
of hydrazine unit. Compound 2a containing the methyl
substituent in position 4 of the thiazole ring exhibits the
highest cytotoxicity against both cell lines. Compound 2b
which contains a chloromethyl and 2c which bears a phenyl
group exhibit lower toxicity against tumor cells, but the
effect is somehow enhanced for their acetylated analogues
3b and 3c. The compound 2d containing a methyl substituent
in position 4 and an acetyl group in position 5 of the thiazole
ring does not exhibit a notable antiproliferative activity upon
the studied cell lines, and the introduction of acetyl group in
its analogue 3d slightly increases this biological effect.
Among the four structures containing ethoxycarbonyl
groups (2e,f and 3e,g), 2f has a notable activity against
Hep2G cell lines, while 3e and 3g, bearing two bulky groups
with high steric hindrance, are characterized by a reduced
biological activity.
All a-halocarbonyl-derivatives were purchased from Sigma–
Aldrich. All reactions as well as column chromatography were
followed by TLC using Merck pre-coated silica gel 60 F254
aluminium sheets. Column chromatography was performed
with Merck silica gel 60 (63–200 mesh).
10-Ethyl-10H-phenothiazine-3-carbaldehyde with m.p. ¼ 988C,
was prepared according to described literature procedures [35].
1-((10-Ethyl-10H-phenothiazin-3-yl)methylidene)-
thiosemicarbazide (1)
(a) To a solution of 10-ethyl-10H-phenothiazine-3-carbaldehyde
(250 mg, 1 mmol) in ethanol (10 mL), thiosemicarbazide
(90 mg, 1 mmol) was added in one portion. The reaction
mixture was heated to reflux for 4 h. The solution was cooled
to room temperature and then the solvent was evaporated
in vacuum. The residue was separated by column chroma-
tography on silica gel (silica gel 60, mesh 0.063–0.2 mm),
by eluting with hexane/acetone (2:1 v/v) or toluene/EtOAc
(1:1 v/v) to afford 270 mg (86%) of the title compound as
yellow crystals with m.p. 170–1728C;
(b) The reaction mixture containing 10-ethyl-10H-phenothia-
zine-3-carbaldehyde (255 mg, 1 mmol) ethanol (5 mL) and
thiosemicarbazide (90 mg, 1 mmol), was introduced in the
quartz reaction vessel, which was sealed and then subjected
to MW irradiation for 30 min at internal temperature of
1008C (Table 1). The product was filtered. The residue was
purified by recrystallization from ethanol and afforded
310 mg (95%) of ith m.p. 1718C.
Conclusions
New phenothiazinyl-thiazolyl-hydrazine derivatives were
successfully prepared by Hantzsch cyclization of 1-(10-ethyl-
10H-phenothiazin-3-yl)-methylidene-thiosemicarbazide with
a-halocarbonyl compounds under microwave irradiation
in sealed reaction vessels. This technique affords excellent
reaction yields in much shorter reaction times as compared
to classical reaction conditions and thus it may be recom-
mended for laboratory scale preparation of compounds 1, 2.
The novel synthesized phenothiazinyl-thiazolyl-hydrazine
derivatives exhibit in vitro antiproliferative activity against
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
IR (ATR) nmax (cmꢀ1): 3425, 3276 (nNH2), 3163 (nNH), 3045 (nCH),
–CH2Cl), 6.80 (d, 1H, 3J ¼ 8.6 Hz, H1), 6.84 (s, 1H, Th–H5), 6.86 (d,
1H, 3J ¼ 8.6 Hz, H9), 6.93 (t, 1H, 3J ¼ 7.3 Hz, H7), 7.09 (dd, 1H,
þ
–
–
–
1597 (nC C), 1543 (nC C), 1247(nC S). MS (EI) m/z 328 (M ), 311,
–
–
–
1
225 (100%), 198; H NMR (400 MHz, CDCl3) d (ppm): 1.43 (t, 3H,
–CH3), 3.94 (q, 2H, N–CH2), 6.82 (d, 1H, H1), 6.85 (d, 1H, H9), 6.93 (t,
1H, H7), 7.09 (dd, 1H, H6), 7.15 (t, 1H, H8), 7.33 (dd, 1H, H2), 7.42 (s,
3J ¼ 7.3 Hz, H6), 7.14 (t, 1H, 3J ¼ 8.6 Hz, H8), 7.35 (dd, 1H,
3
–
J ¼ 8.6 Hz, H ), 7.42 (s, 1H, H ), 7.93 (s, 1H, –CH N), 8.91 (s,
–
2
4
1H, –NH). 13C NMR (100 MHz, DMSO-d6) d (ppm): 13.01 (–CH3),
40.12 (–CH2), 43.39 (–CH2Cl), 109.00 (Th–C5), 114.31 (C9), 115.25
(C1), 122.10 (C7), 123.52 (C4a), 124.64 (C5a), 125.15 (C2), 126.31 (C8),
127.25 (C6), 128.00 (C4), 128.20 (C3), 132.21 (Th–C4), 140.73
1H, H ), 7.68 (s, 1H, –CH N), 9.34 (s, 3H, NH, NH ). 13C NMR
–
–
4
2
(100 MHz, CDCl3) d (ppm): 12.86 (–CH3), 42.13 (–CH2), 114.69 (C9),
115.31 (C1), 123.00 (C7), 123.27 (C4a), 124.90 (C5a), 125.36 (C2),
126.89 (C8), 127.41 (C6), 127.50 (C4), 127.60 (C3), 142.77 (–CH),
–
(CH N), 144.11 (C9a), 145.72 (C10a), 168.15 (Th–C2). Anal. Calcd.
–
for C19H17ClN4S2 (%): C, 56.92; H, 4.27; N, 13.97; S 16.00%. Found
(%): C, 56.91; H, 4.12; N, 13.92; S, 16.00.
–
143.71 (C9a), 147.14 (C10a), 206.9 (–C S); Elemental Anal. Calcd.
–
for C16H16N4S2 (%): C, 58.51; H, 4.91; N, 17.06; S, 19.52. Found (%):
C, 58.43; H, 4.62; N, 16.96; S, 19.45.
1-((10-Ethyl-10H-phenothiazin-3-yl)methylidene)-2-(4-
phenylthiazol-2-yl)hydrazine (2c)
General procedure for the synthesis of phenothiazinyl-
thiazolyl-hydrazine derivatives (2a–g)
Purification by column chromatography on silica gel 60, eluent
hexane/ethyl acetate (2:1 v/v) or crystallization from ethanol. Green
crystals, yield A 62% (0.27 g), B 99%, m.p. ¼ 201–2028C; IR (ATR)
(a) To a solution of thiosemicarbazone 1 (330 mg, 1 mmol) in a
mixture of ethanol (5 mL) and DMF (2 mL), the appropriate a-
halocarbonyl derivative (1 mmol) was added at room
temperature. The reaction mixture was stirred at ambient
temperature for 24 h, then poured on ice and neutralized
with NaHCO3 and the resulting precipitate was collected by
suction filtration. The product was purified by crystallization
or column chromatography.
(b) To a solution of thiosemicarbazone 1 (330 mg, 1 mmol) in
DMF (5 mL), the appropriate a-halocarbonyl derivative
(1 mmol) was added and the obtained reaction mixture
was introduced in the reaction vessel which was sealed
and then subjected to MW irradiation according to con-
ditions presented in Table 2. The reaction product was
poured on ice and then treated as presented above.
nmax (cmꢀ1): 3215 (nNH), 3020 (nC–H), 1684 (nC N), 1600 (nC C),
–
–
–
–
þ
–
–
–
1575 (nC C), 1496 (nC C). MS (EI) m/z: 428 (M ), 399, 252, 239, 226,
–
223 (100%), 176, 134. 1H NMR (400 MHz, CDCl3) d (ppm): 1.41 (t, 3H,
3J ¼ 7.1 Hz, –CH3), 3.92 (q, 2H, 3J ¼ 7.1 Hz, N–CH2), 6.76 (d, 1H,
3J ¼ 8.6 Hz, H1), 6.86 (d, 1H, 3J ¼ 8.6 Hz, H9), 6.93 (t, 1H,
3J ¼ 7.3 Hz, H7), 7.09 (s, 1H, Th–H5), 7.15 (dd, 1H,
3J ¼ 7.3 Hz, H6), 7.25 (t, 1H, 3J ¼ 8.6 Hz, H8), 7.35 (dd, 1H,
3J ¼ 8.6 Hz, H2), 7.42 (s, 1H, H4), 7.60–7.72 (m, 5H, Ar–H), 8.76 (s,
1H, –CH N), 9.78 (s, 1H, –NH). 13C NMR (100 MHz, CDCl3) d (ppm):
–
–
12.90 (–CH3), 42.02 (–CH2), 103 (Th–C5), 114.31 (C9), 115.25 (C1),
122.1 (C7), 123.52 (C4a), 124.64 (C5a), 125.15 (C2, Cmeta), 126.31
(C8, Corto), 126.41 (Cpara), 127.25 (C6), 127.91 (Th–C4), 128 (C4)
–
128.2 (C ), 142.36 (–CH N), 144.08 (C ), 146.08 (C10a), 169.3
–
(Th–C2). Anal. Calcd. for C24H20N4S2 (%): C, 67.26; H, 4.70; N,
3
9a
13.07; S, 14.96. Found (%): C, 67.18; H, 4.63; N, 13.02; S, 14.82.
1-((10-Ethyl-10H-phenothiazin-3-yl)methylidene)-2-(4-
methylthiazol-2-yl)hydrazine (2a)
5-Acetyl-((10-methyl-10H-phenothiazin-3-yl)methylidene)-
2-(4-methylthiazol-2-yl)hydrazine (2d)
Purification by column chromatography on silica gel 60,
toluene/ethyl acetate (2:1 v/v) or crystallization from ethanol.
Yellow crystals, yield A 63% (0.26 g), B 89%, m.p. ¼ 235–2368C; IR
(ATR) nmax (cmꢀ1): 3265 (nNH), 3053 (nC–H), 2987 (nC–H), 2956
Purification by column chromatography on silica gel 60, eluent
hexane/ethyl acetate (2:1 v/v). Yellow-green crystals, yield A 57%
(0.21 g), B 98%, m.p. ¼ 149–1518C; IR (ATR) nmax (cmꢀ1): 31þ65
–
–
–
(nNH), 3055 (nCH), 1576 (nC C), 1496 (nC C). MS (EI) m/z: 366 (M ),
–
252, 239, 226, 223 (100%), 197, 114. 1H NMR (400 MHz, CDCl3)
d (ppm): 1.42 (t, 3H, 3J ¼ 7.1 Hz, –CH3), 2.26 (s, 3H, Th–CH3), 3.95
(q, 2H, 3J ¼ 7.1 Hz, N–CH2), 6.16 (s, 1H, Th–H5), 6.80 (d, 1H,
3J ¼ 8.6 Hz, H1), 6.86 (d, 1H, 3J ¼ 8.6 Hz, H9), 6.93 (t, 1H,
3J ¼ 7.3 Hz, H7), 7.09 (dd, 1H, 3J ¼ 7.3 Hz, H6), 7.14 (t, 1H,
3J ¼ 8.6 Hz, H8), 7.35 (dd, 1H, 3J ¼ 8.6 Hz, H2), 7.42 (s, 1H, H4),
–
–
–
–
(nC–H), 1688 (nC O), 1651 (nC N), 1579 (nC C), 1495 (nC C),
–
–
–
–
1370. MS (EI) m/z: 408 (Mþ), 332, 286, 268, 252, 239, 225,
223(100%), 197, 141. 1H NMR (400 MHz, CDCl3) d (ppm): 1.43
(t, 3H, 3J ¼ 7.1 Hz, –CH3), 2.48 (s, 3H, Th–CH3), 2.59 (s, 3H,
–CO–CH3), 3.95 (q, 2H, 3J ¼ 7.1 Hz, N–CH2), 6.84 (d, 1H,
3J ¼ 8.6 Hz, H1), 6.87 (d, 1H, 3J ¼ 7.6 Hz, H9), 6.93 (t, 1H,
3J ¼ 7.6 Hz, H8), 7.12 (dd, 1H, 3J ¼ 7.6 Hz, H6), 7.15 (t, 1H,
3J ¼ 7.6 Hz, H7), 7.39 (dd, 1H, 3J ¼ 8.6 Hz, H2), 7.45 (s, 1H, H4),
7.76 (s, 1H, –CH N), 8.87 (s, 1H, –NH). 13C NMR (100 MHz, CDCl3)
–
–
d (ppm): 13.01 (–CH3), 16.8 (Th–CH3), 42.03 (–CH2), 102 (Th–C5),
114.64 (C9), 115.15 (C1), 122 (C7), 123.59 (C4a), 124.62 (C5a), 125.05
(C2), 126.50 (C8), 127.35 (C6), 128.00 (C4), 128.20 (C3), 142.13
–
7.76 (s, 1H, –CH N), 8.83 (s, 1H, –NH). 13C NMR (100 MHz, CDCl3)
–
–
(Th–C4), 144.10 (C9a), 145.85 (C10a), 146.09 (CH N), 169.05
–
d (ppm): 12.9 (–CH3), 17.62 (Th–CH3), 29.07 (–CO–CH3), 42.02
(–CH2), 111.63 (Th–C5), 114.31 (C9), 115.25 (C1), 122.10 (C7),
123.52 (C4a), 124.64 (C5a), 125.15 (C2), 126.31 (C8), 127.25 (C6),
(Th–C2). Anal. Calcd. for C19H18N4S2 (%): C, 62.27; H, 4.95;
N 15.29; S 17.50. Found (%): C, 62.18; H, 4.73; N, 15.12; S, 17.41.
–
127.86 (Th–C ), 128.00 (C ), 128.20 (C ), 141.00 (–CH N), 144.08
–
–
(C9a), 146.08 (C10a), 170.00 (Th–C ), 189.70 (C O). Anal. Calcd.
4
4
3
1-((10-Ethyl-10H-phenothiazin-3-yl)methylidene)-2-(4-
chloromethylthiazol-2-yl)hydrazine (2b)
–
for C21H20N4OS2 (%): C, 61.74; H, 4.93; N, 13.71; S, 15.70.
2
Purification by column chromatography on silica gel 60, eluent
hexane/ethylacetate (1:1 v/v). Yellow-brown crystals, yield A 43%
(0.18 g), B 92%, m.p. ¼ 237–2398C; IR (ATR) nmax (cmꢀ1): 3165
Found (%): C, 61.65; H, 4.89; N, 13.64; S, 15.62.
5-Ethoxycarbonyl-((10-methyl-10H-phenothiazin-3-
–
–
–
(nNH), 3050 (nCH), 2920 (nCH), 1599 (nC C), 1559 (nC C), 1497
–
þ
–
yl)methylidene)-2-(4-methylthiazol-2-yl)hydrazine (2e)
Purification by column chromatography on silica gel 60, hexane/
ethyl acetate (1:1 v/v) or crystallization from ethanol. Yellow-
(nC C), 708 (nC–Cl). MS (EI) m/z: 400 (M ), 269, 252, 237, 225, 223
–
(100%), 169, 147. 1H NMR (400 MHz, DMSO-d6) d (ppm): 1.42 (t, 3H,
3J ¼ 7.1 Hz, –CH3), 3.95 (q, 2H, 3J ¼ 7.1 Hz, N–CH2), 4.62 (s, 2H,
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Novel Phenothiazinyl-Thiazolyl-Hydrazine Derivatives
7
–
green crystals, yield A 60% (0.26 g), B 92%, m.p. ¼ 219–2208C; IR
(ATR) nmax (cmꢀ1): 3285 (nNH), 3021 (nC–H), 2952, 2850 (nC–H),
127.38 (C ), 128.09 (C ) 128.20 (C ), 142.21 (–CH N), 144.05 (C9a),
6
–
4
3
146.22 (C10a), 161.64 (Th–C2), 168.49 (–COO). Anal. Calcd.
for C21H20N4O2S2 (%): C, 59.41; H, 4.75; N, 13.20; S, 15.10.
Found (%): C, 59.35; H, 4.69; N, 13.11; S, 15.01.
–
–
–
–
1662 (nC O), 1599 (nC N), 1566 (nC C), 1523 (nC C), 1494
þ
–
–
–
–
–
–
(nC C), 1244, 1094 (nC–O). MS (EI) m/z: 438 (M ), 388, 342, 282,
268 (100%), 252, 239, 199, 149, 119, 97. 1H NMR (400 MHz, DMSO-
d6) d (ppm): 1.37 (t, 3H, 3J ¼ 7.3 Hz, –CH3), 1.43 (t, 3H, 3J ¼ 7.1 Hz,
–CH3), 2.59 (s, 3H, Th–CH3), 3.94 (q, 2H, 3J ¼ 7.1 Hz, N–CH2), 4.31
(q, 2H, 3J ¼ 7.3 Hz, O–CH2), 6.83 (d, 1H, 3J ¼ 8.6 Hz, H1), 6.89 (d,
1H, 3J ¼ 7.6 Hz, H9), 6.92 (t, 1H, 3J ¼ 7.6 Hz, H7), 7.11 (dd, 1H,
General procedure for acetylation of 2a–g
One millimole of phenothiazinyl-thiazolyl-hydrazine derivative
(2a–g) was treated with 5 mL of acetic anhydride and catalytic
amounts of pyridine. The resulting mixture was heated for
15 min to 1308C under stirring, then the acetic anhydride was
evaporated under reduced pressure. The obtained solid was
purified by recrystallization or column chromatography.
3J ¼ 7.6 Hz, H6), 7.16 (t, 1H, 3J ¼ 7.6 Hz, H8), 7.37 (dd, 1H,
3
–
–
J ¼ 8.6 Hz, H ), 7.45 (s, 1H, H ), 7.79 (s, 1H, CH N), 9.14 (s,
2
4
1H, –NH). 13C NMR (100 MHz, DMSO-d6) d (ppm): 12.84 (–CH3),
14.41 (–CH3), 16.62 (Th–CH3), 42.11 (–CH2), 60.87 (–CH2), 112.33
(Th–C5), 114.31 (C9), 115.25 (C1), 122.10 (C7), 123.52 (C4a), 124.64
(C5a), 124.71 (Th–C4), 125.15 (C2), 126.31 (C8), 127.25 (C6), 128.00
–
N0-((10-Methyl-10H-phenothiazin-3-yl)methylene)-N-
(4-methylthiazol-2-yl)acetohydrazide (3a)
(C ) 128.20 (C ), 143.00 (–CH N), 144.58 (C9a), 146.80 (C10a),
–
4
3
162.20 (Th–C2), 168.93 (–COO). Anal. Calcd. for C22H22N4O2S2
(%): C, 60.25; H, 5.06; N, 12.78; S, 14.62. Found (%): C, 59.95; H,
4.99; N, 12.61; S, 14.61.
Purification by column chromatography on silica gel 60, toluene/
ethyl acetate (1:1 v/v). Yellow crystals, yield 58.2% (0.20 g),
m.p. ¼ 73–758C; IR (ATR) nmax (cmꢀ1): 3054, 2970, 2850 (nCH),
–
–
–
–
1698 (nC O), 1587 (nC C), 1453 (nC C), 1363 (–COCH ). MS (EI)
–
–
3
m/z: 408 (Mþ), 366, 252, 239, 226, 223, 197, 114, 43 (100%). 1H NMR
Ethyl 2-(2-(2-((10-methyl-10H-phenothiazin-3-yl)-
methylene)hydrazinyl)thiazol-4-yl)acetate (2f)
3
(400 MHz, CDCl3) d (ppm): 1.43 (t, 3H, J ¼ 7.1 Hz, –CH3), 2.46 (s,
3H, –CH3), 2.48 (s, 3H, –CH3), 3.95 (q, 2H, 3J ¼ 7.1 Hz, N–CH2), 6.68
(s, 1H, Th–H5), 6.81 (d, 1H, 3J ¼ 8.6 Hz, H1), 6.89 (d, 1H,
3J ¼ 8.6 Hz, H9), 7.01 (t, 1H, 3J ¼ 7.3 Hz, H7), 7.12 (dd, 1H,
Purification by column chromatography on silica gel 60, hexane/
ethyl acetate (1:1 v/v). Green crystals, yield A 68% (0.29 g), B
94%, m.p. ¼ 220–2218C; IR (ATR) nmax (cmꢀ1): 3243 (nNH), 3030
3J ¼ 7.3 Hz, H6), 7.16 ppm (t, 1H, 3J ¼ 8.6 Hz, H8), 7.38 (dd, 1H,
–
–
–
(nC–H), 2956 (nC–H), 2852 (nC–H), 1722 (nC O), 1596 (nC C), 1237,
3
J ¼ 8.6 Hz, H ), 7.49 (s, 1H, H ), 8.21 (s, 1H, –CH N). 13C NMR
–
–
–
2
4
1024 (nC–O). MS (EI) m/z: 438 (Mþ), 365, 352, 268 (100%), 252, 226,
199, 149, 119, 97, 73. 1H NMR (400 MHz, CDCl3) d (ppm): 1.28 (t,
3H, 3J ¼ 7.3 Hz, –CH3), 1.42 (t, 3H, 3J ¼ 7.1 Hz, –CH3), 3.58 (s, 2H,
Th–CH2–COO), 3.94 (q, 2H, 3J ¼ 7.1 Hz, N–CH2), 4.19 (q, 2H,
3J ¼ 7.3 Hz, O–CH2), 6.44 (s, 1H, Th–H5), 6.82 (d, 1H,
3J ¼ 8.6 Hz, H1), 6.86 (d, 1H, 3J ¼ 8.1 Hz, H9), 6.92 (t, 1H,
3J ¼ 7.6 Hz, H7), 7.11 (dd, 1H, 3J ¼ 7.6 Hz, H6), 7.14 ppm (t, 1H,
3J ¼ 8.1 Hz, H8), 7.36 (dd, 1H, 3J ¼ 8.6 Hz, H2), 7.44 (s, 1H, H4),
(100 MHz, CDCl3) d (ppm): 13.01 (–CH3), 16.8 (Th–CH3), 22.36
(–CH3), 42.03 (–CH2), 106.00 (Th–C5), 114.64 (C9), 115.45 (C1),
123.00 (C7), 123.59 (C4a), 124.68 (C5a), 125.05 (C2), 126.55 (C8),
127.65 (C6), 128.00 (C4), 128.20 (C3), 143.16 (Th–C4), 145.22 (C9a),
–
–
146.03 (C10a), 147.17 (CH N), 167.23 (Th–C ), 169.38 (C O). Anal.
2
–
–
Calcd. for C21H20N4OS2 (%): C, 61.74; H, 4.93; N, 13.71; S, 15.70.
Found (%): C, 61.64; H, 4.91; N, 13.71; S, 15.66.
7.76 (s, 1H, –CH N), 9.78 (s, 1H, –NH). 13C NMR (100 MHz, CDCl3)
–
–
N-(4-(Chloromethyl)thiazol-2-yl)-N0-((10-methyl-10H-
phenothiazin-3-yl)methylene)acetohydrazide (3b)
Purification by column chromatography on silica gel 60, hexane/
ethyl acetate (1:2, v/v). Yellow-brown crystals yield 49.3%
(0.19 g), m.p. ¼ 116–1178C; IR (ATR) nmax (cmꢀ1): 3058 (nC–H),
d (ppm): 12.90 (–CH3), 14.12 (–CH3), 36.15 (–CH2), 42.06 (–CH2),
61.26 (–CH2), 105.23 (Th–C5), 114.71 (C9), 115.17 (C1), 122.74 (C7),
125.06 (C4a), 126.79 (C5a), 126.93 (Th–C4), 127.31 (C2), 127.37 (C8),
–
127.85 (C ), 128.00 (C ), 128.20 (C ), 143.08 (–CH N), 144.03 (C9a),
3
–
6
4
146.28 (C10a), 163 (Th–C2), 169.32 (–COO). Anal. Calcd.
for C22H22N4O2S2 (%): C, 60.25; H, 5.06; N, 12.78; S, 14.62.
Found (%): C, 59.88; H, 4.99; N, 12.71; S, 14.52.
–
–
–
2981 (nC–H), 2934 (nC–H), 2872 (nC–H), 1696 (nC O), 1599 (nC N),
–
þ
–
–
1574 (nC C), 1367. MS (EI) m/z: 443 (M ), 399, 267, 253, 226, 223,
197, 169, 147, 43 (100%). 1H NMR (400 MHz, DMSO-d6) d (ppm):
1.44 (t, 3H, 3J ¼ 7.1 Hz, –CH3), 2.55 (s, 3H, –CH3), 3.97 (q, 2H,
3J ¼ 7.1 Hz, N–CH2), 4.64 (s, 2H, –CH2Cl), 6.87 (d, 1H,
3J ¼ 8.6 Hz, H1), 6.71 (s, 1H, Th–H5), 6.93 (d, 1H,
3J ¼ 8.6 Hz, H9), 6.98 (t, 1H, 3J ¼ 7.3 Hz, H7), 7.16 (dd, 1H,
4-Ethoxycarbonyl-((10-methyl-10H-phenothiazin-3-yl)-
methylidene)-2-(thiazol-2-yl)hydrazine (2g)
Purification by column chromatography on silica gel 60, hexane/
acetone (1:1 v/v) or crystallization from ethanol. Yellow crystals,
yield A 59% (0.25 g), B 91%, m.p. ¼ 204–2058C; IR (ATR) nmax
(cmꢀ1): 3221 (nNH), 3135 (nC–H), 3079 (nC–H), 2978 (nC–H),
3J ¼ 7.3 Hz, H6), 7.20 (t, 1H, 3J ¼ 8.6 Hz, H8), 7.56 (s, 1H, H4),
7.63 (dd, 1H, J ¼ 8.6 Hz, H ), 9.78 (s, 1H, –CH N). 13C NMR
3
–
–
2
(100 MHz, DMSO-d6) d (ppm): 13.01 (–CH3), 22.32 (–CH3), 41.22
(–CH2), 43.39 (–CH2Cl), 112 (Th–C5), 114.32 (C9), 115.65 (C1),
122.12 (C7), 123.52 (C4a), 124.64 (C5a), 125.15 (C2), 126.31 (C8),
–
–
2935 (nC–H), 2867 (nC–H), 1681 (nC O), 1577 (nC C), 1493
þ
–
–
–
–
(nC C), 1241, 1095 (nC–O). MS (EI) m/z: 424 (M ), 268, 252, 223
(100%), 199, 156, 149, 73. 1H NMR (400 MHz, CDCl3) d (ppm): 1.37
(t, 3H, 3J ¼ 7.1 Hz, –CH3), 1.42 ppm (t, 3H, 3J ¼ 6.8 Hz, –CH3),
3.93 (q, 2H, 3J ¼ 6.8 Hz, N–CH2), 4.36 (q, 2H, 3J ¼ 7.1 Hz, O–CH2),
6.83 (d, 1H, 3J ¼ 8.6 Hz, H1), 6.87 (d, 1H, 3J ¼ 7.6 Hz, H9), 6.92 (t,
1H, 3J ¼ 7.8 Hz, H7), 7.12 (dd, 1H, 3J ¼ 7.8 Hz, H6), 7.15 (t, 1H,
3J ¼ 7.6 Hz, H8), 7.36 (dd, 1H, 3J ¼ 8.6 Hz, H2), 7.43 (s, 1H, H4),
127.25 (C6), 129.47 (C4), 129.65 (C3), 145.23 (Th–C4), 145.75 (C9a)
–
,
146.02 (C10a), 147.73 (CH N), 162.34 (Th–C ), 169.17 (C O). Anal.
–
–
Calcd. for C21H19ClN4OS2 (%): C, 56.94; H, 4.32; N, 12.65; S, 14.47.
–
2
Found (%): C, 56.71; H, 4.27; N, 12.54; S, 14.42.
N0-((10-Ethyl-10H-phenothiazin-3-yl)methylene)-N-
(4-phenylthiazol-2-yl) acetohydrazide (3c)
Purification by column chromatography on silica gel 60, toluene/
ethyl acetate (1:1 v/v) or crystallization from ethanol. Green
7.57 (s, 1H, Th–H ), 7.76 (s, 1H, –CH N), 9.17 (s, 1H, –NH). 13C NMR
–
–
5
(100 MHz, CDCl3) d (ppm): 12.90 (–CH3), 14.34 (–CH3), 42.05
(–CH2), 61.25 (–CH2), 114.74 (C9), 115.18 (C1), 118.48 (C7),
122.75 (Th–C5), 123 (C4a), 124.69 (C5a), 125.38 (C2), 126.56 (C8),
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
A. Ignat et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
crystals, yield 65% (0.30 g), m.p. ¼ 170–1728C; IR (ATR) nmax (cmꢀ1):
125.15 (C2), 126.51 (C8), 127.45 (C6), 128.24 (C4), 130.12 (C3), 144.86
–
–
–
3107 (nC–H), 3057 (nC–H), 2985 (nC–H), 1679 (nC O), 1599 (nC N),
–
(C9a), 146.12 (C10a), 146.32 (–CH N), 169.53 (Th–C ), 188.05 (–CO),
–
190.02 (–COO). Anal. Calcd. for C24H24N4O3S2 (%): C, 59.98; H, 5.03;
–
1575 (nC C), 1548 (nC C), 1362. MS (EI) m/z: 470 (M ), 427, 398, 267,
–
2
þ
–
–
–
252, 239, 226, 223, 176, 134, 43 (100%). 1H NMR (400 MHz, CDCl3)
N, 11.66; S, 13.34. Found (%): C, 59.85; H, 4.99; N, 11.58; S, 13.28.
3
d (ppm): 1.44 (t, 3H, J ¼ 7.1 Hz, –CH3), 2.59 (s, 3H, –CH3), 3.96 (q,
3
3
2H, J ¼ 7.1 Hz, N–CH2), 6.88 (d, 1H, J ¼ 8.6 Hz, H1), 6.90 (d, 1H,
Ethyl 2-(2-(1-acetyl-2-((10-ethyl-10H-phenothiazin-3-yl)-
methylene)hydrazinyl)-thiazol-4-yl)-acetate (3f)
3J ¼ 8.6 Hz, H9), 6.95 (t, 1H, 3J ¼ 7.3 Hz, H7), 7.09 (dd, 1H,
3J ¼ 7.3 Hz, H6), 7.15 (t, 1H, J ¼ 8.6 Hz, H8), 7.21 (s, 1H, Th–H5),
3
Purification by column chromatography on silica gel 60,
toluene/ethyl acetate (1:1, v/v). Yellow crystals, yield 54.8%
(0.26 g), m.p. ¼ 169–1708C; IR (ATR) nmax (cmꢀ1): 3080 (nC–H),
7.41–7.43 (m, 5H, Ar–H), 7.57 (s, 1H, H4), 7.62 (dd, 1H,
–
J ¼ 8.6 Hz, H ), 9.78 (s, 1H, –CH N). 13C NMR (100 MHz, CDCl3)
3
–
2
d (ppm): 12.85 (–CH3), 22.86 (–CH3), 42.45 (–CH2), 111.7 (Th–C5),
114.73 (C9), 115.56 (C1), 122.98 (C7), 123.22 (C4a), 123.53 (C5a), 126
(C2), 126.44 (Cmeta), 127.4 (C8), 127.57 (Corto), 128.00 (Cpara), 128.15
–
–
–
2970 (nC–H), 2860 (nC–H), 1740 (nC O ester), 1680 (nC O ketone),
–
–
–
–
–
–
–
–
1590 (nC N), 1575 (nC C), 1544 (nC C), 1496 (nC C), 1362, 1285
–
(nC–O), 1080 (nC–O). MS (EI) m/z: 480 (Mþ), 437, 351, 268, 252
(100%), 226, 198, 185, 199, 170, 149, 119, 97, 43. 1H NMR
(400 MHz, CDCl3) d (ppm): 1.32 (t, 3H, 3J ¼ 7.3 Hz, –CH3), 1.43
(t, 3H, 3J ¼ 7.1 Hz, –CH3), 2.64 (s, 3H, –CH3), 3.83 (s, 2H,
Th–CH2–COO), 3.94 (q, 2H, 3J ¼ 7.1 Hz, N–CH2), 4.29 (q, 2H,
3J ¼ 7.3 Hz, O–CH2), 6.83 (d, 1H, 3J ¼ 8.6 Hz, H1), 6.89 (d, 1H,
3J ¼ 8.1 Hz, H9), 6.96 (t, 1H, 3J ¼ 7.6 Hz, H7), 7.12 (dd, 1H,
3J ¼ 7.6 Hz, H6), 7.15 (t, 1H, 3J ¼ 8.1 Hz, H8), 7.53 (s, 1H, H4),
7.61 (dd, 1H, 3J ¼ 8.6 Hz, H2), 7.87 (s, 1H, Th–H5), 9.73 (s, 1H,
–
(C ), 128.24 (Th–C ), 128.70 (C ), 128.77 (C ), 130.17 (CH N), 143.05
–
–
(C9a), 150.87 (C10a), 172.18 (Th–C ), 190.06 (C O). Anal. Calcd.
6
4
4
3
–
for C26H22N4OS2 (%): C, 66.36; H, 4.71; N, 11.91; S, 13.62. Found
2
(%): C, 66.28; H, 4.62; N, 11.91; S, 13.54.
0
N-(5-Acetyl-4-methylthiazol-2-yl)-N -((10-ethyl-10H-
phenothiazin-3-yl)methylene)acetohydrazide (3d)
Purification by column chromatography on silica gel 60,
toluene/ethyl acetate (1:1 v/v) or crystallization from ethanol.
Yellow crystals, yield 52.4% (0.26 g), m.p. ¼ 173–1748C; IR (ATR)
nmax (cmꢀ1): 3058 (nC–H), 2969 (nC–H), 2928 (nC–H), 2860 (nC–H),
–CH N). 13C NMR (100 MHz, CDCl3) d (ppm): 12.91 (–CH3), 14.32
–
–
(–CH3), 29.68 (–CH3), 36.25 (–CH2), 42.36 (–CH2), 61.26 (–CH2),
105.23 (Th–C5), 114.82 (C9), 115.27 (C1), 122.83 (C7), 125.16
(C4a), 126.80 (C5a), 126.95 (Th–C4), 127.33 (C2), 127.39 (C8),
127.86 (C6), 128.05 (C4), 129.22 (C3), 144.73 (C9a), 146.32 (C10a),
–
–
–
–
1681 (nC O), 1657 (nC O), 1597 (nC N), 1572 (nC C), 1502
þ
–
–
–
–
–
–
(nC C), 1365, 1328. MS (EI) m/z: 450 (M ), 407, 286, 268, 252,
239, 225, 223, 197, 141, 43 (100%). 1H NMR (400 MHz, CDCl3)
d (ppm): 1.44 (t, 3H, 3J ¼ 7.1 Hz, –CH3), 2.51 (s, 3H, Th–CH3), 2.62
(s, 3H, –CH3), 2.65 (s, 3H, –CH3), 3.97 (q, 2H, 3J ¼ 7.1 Hz, N–CH2),
6.89 (d, 1H, 3J ¼ 7.6 Hz, H9), 6.90 (d, 1H, 3J ¼ 8.6 Hz, H1), 6.95 (t,
1H, 3J ¼ 7.6 Hz, H8), 7.09 (dd, 1H, 3J ¼ 7.6 Hz, H6), 7.15 (t, 1H,
3J ¼ 7.6 Hz, H7), 7.57 (s, 1H, H4), 7.63 (dd, 1H, 3J ¼ 8.6 Hz, H2),
146.38 (–CH N), 169.26 (Th–C ), 188.12 (–CO), 190.07 (–COO).
2
–
–
Anal. Calcd. for C24H24N4O3S2 (%): C, 59.98; H, 5.03; N, 11.66;
S, 13.34. Found (%): C, 59.85; H, 4.98; N, 11.63; S, 13.28.
Ethyl 2-(1-acetyl-2-((10-ethyl-10H-phenothiazin-3-yl)-
methylene)hydrazinyl)thiazole-4-carboxylate (3g)
9.78 (s, 1H, –CH N). 13C NMR (100 MHz, CDCl3) d (ppm): 13.00
–
–
Purification by column chromatography on silica gel 60,
toluene/acetone (1:2 v/v). Yellow crystals, yield 60.5%
(0.28 g), m.p. ¼ 114–1158C; IR (ATR) nmax (cmꢀ1): 3056 (nC–H),
(–CH3), 17.69 (Th–CH3), 29.07 (–CO–CH3), 31.56 (–CO–CH3), 42.60
(–CH2), 111.93 (Th–C5), 114.51 (C9), 115.35 (C1), 122.58 (C7), 123.82
(C4a), 124.94 (C5a), 125.45 (C2), 126.85 (C8), 127.69 (C6), 128.00 (C4),
128.20 (C3), 141.83 (Th–C4), 144.08 (C9a), 146.08 (C10a), 145.31
–
2982 (nC–H), 2938 (nC–H), 2906 (nC–H), 1730 (nC O ester), 1671
–
–
–
–
–
–
–
–
–
(nC O ketone), 1600 (nC N), 1575 (nC C), 1548 (nC C), 1362,
–
–
–
(–CH N), 169.32 (Th–C ), 188.72 (–C O), 189.31 (–C O). Anal.
2
–
–
–
1203 (nC–O ester), 1070 (nC–O ester). MS (EI) m/z: 466 (Mþ),
423, 350, 268, 252, 223, 199, 156, 149, 73, 43 (100%). 1H NMR
(400 MHz, CDCl3) d (ppm): 1.38 (t, 3H, 3J ¼ 7.1 Hz, –CH3), 1.43 (t,
3H, 3J ¼ 6.8 Hz, –CH3), 2.65 (s, 3H, –CH3), 3.94 (q, 2H, 3J ¼ 6.8 Hz,
N–CH2), 4.38 (q, 2H, 3J ¼ 7.1 Hz, O–CH2), 6.84 (d, 1H,
3J ¼ 8.6 Hz, H1), 6.89 (d, 1H, 3J ¼ 7.6 Hz, H9), 6.96 (t, 1H,
3J ¼ 7.8 Hz, H7), 7.13 (dd, 1H, 3J ¼ 7.8 Hz, H6), 7.16 (t, 1H,
3J ¼ 7.6 Hz, H8), 7.49 (s, 1H, H4), 7.58 (dd, 1H, 3J ¼ 8.6 Hz, H2),
Calcd. for C23H22N4O2S2 (%): C, 61.31; H, 4.92; N, 12.43; S,
14.23. Found (%): C, 61.22; H, 4.91; N, 12.36; S, 14.18.
Ethyl 2-(1-acetyl-2-((10-ethyl-10H-phenothiazin-3-
yl)methylene)hydrazinyl)-4-methylthiazole-5-carboxylate
(3e)
Purification by column chromatography on silica gel 60, toluene/
ethyl acetate (1:2 v/v) or crystallization from ethanol. Yellow-green
crystals, yield 62.6% (0.29 g), m.p. ¼ 163–1648C; IR (ATR) nmax
(cmꢀ1): 3050 (nC–H), 2987 (nC–H), 2956 (nC–H), 2902 (nC–H),
7.89 (s, 1H, Th–H ), 9.78 (s, 1H, –CH N). 13C NMR (100 MHz,
–
–
5
CDCl3) d (ppm): 12.9 (–CH3), 14.34 (–CH3), 29.66 (–CH3), 42.25
(–CH2), 61.29 (–CH2), 114.84 (C9), 115.28 (C1), 118.49 (C7),
122.85 (Th–C5), 123.65 (C4a), 124.79 (C5a), 126.38 (C2), 126.66
(C8), 127.48 (C6), 128.19 (C4), 128.22 (C3), 144.15 (C9a), 146.23
–
–
–
1707 (nC O ester), 1680 (nC O ketone), 1597 (nC N), 1574
–
–
(nC C), 1544 (nC C), 1496 (nC C), 1362, 1296 (nC–O), 1096
–
–
–
–
–
–
–
(nC–O). MS (EI) m/z: 480 (Mþ), 437, 392, 388, 282, 267, 252, 225,
199, 149, 119, 97, 43 (100%). H NMR (400 MHz, CDCl3) d (ppm):
–
(C10a), 146.36 (–CH N), 169.26 (Th–C ), 188.22 (–CO), 190.17
2
–
1
(–COO). Anal. Calcd. for C23H22N4O3S2 (%): C, 59.21; H, 4.75; N,
12.01; S, 13.74. Found (%): C, 58.83; H, 4.65; N, 11.91; S, 13.71.
1.38 (t, 3H, 3J ¼ 7.3 Hz, –CH3), 1.44 (t, 3H, 3J ¼ 7.1 Hz, –CH3), 2.62
(s, 3H, Th–CH3), 2.65 (s, 3H, –CH3), 3.96 (q, 2H, 3J ¼ 7.1 Hz, N–CH2),
4.33 (q, 2H, 3J ¼ 7.3 Hz, O–CH2), 6.89 (d, 1H, 3J ¼ 8.6 Hz, H1), 6.91
(d, 1H, 3J ¼ 8.6 Hz, H9), 6.96 (t, 1H, 3J ¼ 7.6 Hz, H7), 7.09 (dd, 1H,
Biological activity
In vitro anticancer screening
3J ¼ 7.6 Hz, H6), 7.15 (t, 1H, 3J ¼ 8.6 Hz, H8), 7.56 (s, 1H, H4), 7.63
3
(dd, 1H, J ¼ 8.6 Hz, H ), 9.78 (s, 1H, –CH N). 13C NMR (100 MHz,
–
–
2
Cell culture, treatment and cytotoxicity assessment
Cell cultures were performed using Class II LaminAir laminar
hoods, Uniequip incubator, Heidolph Titramax 1000 small
CDCl3) d (ppm): 12.85 (–CH3), 14.34 (–CH3), 29.68 (–CH3), 32.39
(Th–CH3), 42.44 (–CH2), 60.90 (–CH2), 114.37 (Th–C5), 115.31 (C9),
115.42 (C1), 123.10 (C7), 123.53 (C4a), 123.99 (C5a), 124.45 (Th–C4),
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
Novel Phenothiazinyl-Thiazolyl-Hydrazine Derivatives
9
vibration orbit platform shaker with reproducible temperature
control, Hettich centrifuge with spin-out rotor.
solution was added to each well. After 1 h incubation at 378C,
MTT solution was removed, and formazan crystals were solubil-
ized by adding DMSO, which leads to their release from the living
cells.
The 96-well plates were measured at 492 nm with a multi-
mode microplate reader, by monochromator-based absorbance
detection. The optical density, quantified by colorimetric
measurements, is in direct proportional relationship with the
amount of formazan in the cells [38] and it is an indicator of the
cellular proliferation [39].
Spectrometric measurements were completed with Biotek
Synergy 2 Multi-Mode Microplate Reader with SQ Xenon Flash
light source, by well-area colorimetric scanning.
Experimental results were evaluated with the Graph Pad Prism
5 biostatistics software.
Cell cultures
In order to assess the antiproliferative and the antimitotic prop-
erties of phenothiazine derivatives 1–3, the biological systems we
used were the tumor cell lines: the highly proliferating HepG2
and CC531S cells. The HepG2 human hepatoblastoma tumor
cells were acquired from the European Collection of Cell
Cultures (ECCAC). The CC531S colon adenocarcinoma cell line
The financial support from Romanian Ministry of Education, Research,
Youth and Sports, grant ID PCCE 140 is greatly acknowledged.
The authors have declared no conflict of interest.
was
a generous gift from Dr. Calin Precup from ‘‘Iuliu
Hatieganu’’ University of Medicine and Pharmacy in Cluj
Napoca.
References
Both cell lines are adherent, they were cultivated under sterile
conditions on 25-cm2 Cole-type culture flasks (Nunclon Easy
Flask), in RPMI-1640 cell growth media, supplemented with fetal
calf serum (FCS), Hepes, penicillin–streptomycin and glutamine
(all chemicals from Sigma). Culture flasks were kept in sterile
incubators having constant temperature (378C), CO2 level (5%)
and humidified atmosphere. Passages were performed using
mechanic-enzymatic methods.
[1] G. Aridoss, S. Amirthaganesan, M. S. Kim, J. T. Kim, Y. T.
Jeong, Eur. J. Med. Chem. 2009, 44 (10), 4199–4210.
[2] P. Karegoudar, M. S. Karthikeyan, D. J. Prasad, M. Mahalinga,
B. S. Holla, N. S. Kumari, Eur. J. Med. Chem. 2008, 44 (2), 261–
267.
[3] B. S. Holla, K. V. Malini, B. S. Rao, B. K. Sarojini, N. S. Kumari,
Eur. J. Med. Chem. 2003, 38 (3), 313–318.
[4] M.-H. Shih, Y.-S. Su, C.-L. Wu, Chem. Pharm. Bull. 2007, 55 (8),
1126–1135.
Cell treatment and cytotoxicity evaluation
[5] H. I. El-Subbagh, A. M. Al-Obaid, Eur. J. Med. Chem. 1996, 31
(12), 1017–1021.
We used the colorimetric MTT method to complete the evalu-
ation of biomaterial cytotoxicity. This cellular inhibition test
applicable to the eukaryotic cells is based on the enzymatic
turnover of mitochondrial dehydrogenase. The MTT tetrazolium
salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (Sigma M5655) is a pale yellow water-soluble dye, which
is easily taken up by viable cells and reduced [36, 37] to the purple
formazan product 1-(4,5-dimethyl-1,3-thiazol-2-yl)-3,5-di(phenyl)-
2H-tetrazole. A cleavage of the tetrazolium rings occurs due to
the presence of mitochondrial reductases only in intact living
cells. The insoluble purple formazan product does not cross the
cellular membrane and remains accumulated in the cells. The
crystals are dissolved into a colored solution with organic sol-
vents such dimethyl sulfoxide (DMSO).
[6] H. T. Fahmy, S. A. Rostom, A. A. Bekhit, Arch. Pharm. 2002, 335
(5), 213–222.
[7] A. Andreani, M. Rambaldi, A. Locatelli, R. Bossa, A. Fraccari,
I. Galatulas, J. Pharm. Belg. 1993, 48 (5), 378–382.
[8] L. Becan, E. Wagner, Arzneimittelforsch. Drug. Res. 2008, 58 (10),
521–528.
[9] S. C. Mitchell, Curr. Drug Targets 2006, 7 (9), 1181–1189.
[10] A. D. Mosnaim, V. V. Ranade, M. E. Wolf, J. Puente, M. A.
Valenzuela, Am. J. Ther. 2006, 13 (3), 261–273.
[11] F. Barbieri, A. Alama, B. Tasso, V. Boido, C. Bruzzo, F.
Sparatore, Invest. New Drugs 2003, 21 (4), 413–420.
For the experiment, cells were placed on flat bottom 96-well
microplates, in 200 mL media, and after 24 h of incubation the
compounds 1–3 solutions were added, 10 mL in each well. The 12
compounds 1–3 were solubilized in DMSO, to obtain the stock
solutions and then sequential dilutions were obtained using
phosphate buffered saline solution (PBS-Sigma). In the treated
wells the final concentrations of 1–3 were between 0.1 and 50 mg/
mL for each compound, for every compound a series of 8–10
concentration were tested. For the cell proliferation, the refer-
ence values were the untreated cells. For every compound we
used reagent blank (media and MTT), color control (wells con-
tains media and phenothiazine derivatives 1–3 solution, but no
cells). As positive control we used the antineoplastic drug cispla-
tin (Actavis Sindan Pharma) in the same concentrations as the
studied compounds.
[12] V. K. Tandon, H. K. Maurya, A. Tripathi, G. B. Shiva Keshava,
P. K. Shukla, P. Srivastava, D. Panda, Eur. J. Med. Chem. 2009, 44
(3), 1086–1092.
[13] B. Pajak, J. Molnar, H. Engi, A. Orzechowski, In Vivo 2005, 19
(6), 1101–1104.
[14] I. Gil-Ad, B. Shtaif, Y. Levkovitz, M. Dayag, E. Zeldich, A.
Weizman, J. Mol. Neurosci. 2004, 22 (3), 189–198.
[15] P. Karmakar, A. T. Natarajan, R. K. Poddar, U. B. Dasgupta,
Toxicol. Lett. 2001, 125 (1–3), 19–28.
[16] A. Bisi, M. Meli, S. Gobbi, A. Rampa, M. Tolomeo, L.
Dusonchet, Bioorg. Med. Chem. 2008, 16 (13), 6474–6482.
´
´
´
[17] A. Konya, A. Andor, P. Satorhelyi, K. Nemeth, I. Kurucz,
Biochem. Biophys. Res. Commun. 2006, 346 (1), 45–50.
[18] D. Zaharia, V. Zaharia, D. Matinca, I. Simiti, Farmacia 1999,
47 (2), 51–60.
Three independent experiments were performed, all in trip-
licate. After 24 h of treatment with phenothiazine derivatives 1–
3, the culture media was removed from the wells, without dis-
turbing the attached cells, and 100 mL of MTT-Hanks media
[19] V. Zaharia, L. Vlase, N. Palibroda, Farmacia 2001, 49 (4),
54–61.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

10
A. Ignat et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–10
[20] V. Zaharia, I. Chirtoc, Farmacia 2001, 49 (6), 24–31.
[30] M. Welsch, Ng. Ph. Buu-Hoi, G. Dechamps, N. Hoan,
H. Le Bihan, F. Binon, Compt. Rend. 1951, 232, 1608–1610.
[21] V. Zaharia, D. Zaharia, I. Chirtoc, N. Palibroda, Clujul Med.
2002, 75 (4), 713–719.
[31] G. Cerbani, L. Turbanti, Bull. Chim. Farm. 1968, 108 (7), 433–
439.
[22] V. Zaharia, A. Ignat, N. Palibroda, B. Ngameni, V. Kuete, C. N.
Fokunang, M. L. Moungang, B. T. Ngadjui, Eur. J. Med. Chem.
2010, 45, 5080–5085.
´
[32] I. Kwiecien, H. Rokita, E. Lorenc-Koci, M. Sokolowska,
L. Włodek, Fundam. Clin. Pharmacol. 2007, 21 (1), 95–103.
[23] S. V. Filip, I. A. Silberg, E. Surducan, M. Vlassa, V. Surducan,
Synth. Commun. 1998, 28, 337–341.
[33] Y. Lu, A. Cederbaum, Free Radic. Biol. Med. 2007, 43 (7), 1061–
1075.
[24] D. Porumb, C. Cristea, I. A. Silberg, Studia UBB Ser. Chem. 2002,
47 (1–2), 45–50; L. Gaina, D. Porumb, I. Silaghi-Dumitrescu, C.
Cristea, L. Silaghi-Dumitrescu, Can. J. Chem. 2010, 88 (1), 42–49;
L. Gaina, C. Cristea, C. Moldovan, D. Porumb, E. Surducan, C.
Deleanu, A. Mahamoud, J. Barbe, I. A. Silberg, Int. J. Mol. Sci.
2007, 8, 70–80.
[34] J. H. Velthuis, Z. Gavric, H. J. de Bont, J. F. Nagelkerke,
Biochem. Pharmacol. 2005, 69 (3), 463–471.
´
[35] M. Tosa, Cs. Paizs, C. Majdik, P. Moldovan, L. Novak, P.
´
´
Kolonits, E. Szabo, L. Poppe, F. D. Irimie, J. Mol. Catal. B-
Enzym. 2002, 17, 241–248.
[36] D. M. L. Morgan, Polyamine Protocols, in: Methods in
Molecular Biology, vol. 79, Humana Press, Clifton, NJ, USA
1997, Part VI, pp. 179–184.
[25] M. Mayer, P. T. Lang, S. Gerber, P. B. Madrid, I. G. Pinto, R. K.
Guy, T. L. James, Chem. Biol. 2006, 13, 993–1000.
[26] A. G. Sudhakar, A. P. Amol, B. D. Madhukar, Phosphorous
Sulfur Silicon Relat. Elem. 2010, 185, 103–109.
[37] R. Supino, In vitro toxicity testing protocols, in:
Methods in Molecular Biology, vol. 43 (Ed.: S. O. Hare, C. K.
Atterwill), Humana Press, Clifton, NJ, USA 1995, pp. 137–
149.
[27] L. G. Santosh, S. Hiroki, Tetrahedron 2010, 66, 3314–3317.
[28] D. P. Khrustalev, A. A. Suleimenova, S. D. Fazylov, A. M.
Gazaliev, T. S. Zhivotova, Izv. Nats. Akad. Nauk Resp. Kaz. Ser.
Khim. 2008, 5, 18–21.
[38] T. Mosmann, J. Immunol. Methods 1983, 65 (1–2), 55–563.
[39] J. D. Burton, Chemosensitivity, in: Methods in Molecular
Medicine, vol. 110 (Ed.: R. D. Blumenthal), Humana Press,
Clifton, NJ, USA 2005, pp. 69–78.
[29] Z. Xue, X. Zhang, Y. Tian, Yuanzi Yu Fenzi Wuli Xuebao 2002, 19
(3), 307–312; Chem. Abstr. 138, 287017.
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com